The goal of this proposal is to understand how molecular guidance cues are integrated to direct fasciculating axons in the zebrafish brain. MLF axons defasciculate following knockdown of Sema3D signaling and fail to initially converge and subsequently extend after TAG-1 knockdown. MLF fasciculation will be analyzed following concurrent manipulation of Sema3D signaling molecules to identify critical components involved in regulating fasciculation. Cell adhesion assays will be used to test whether Sema3D modifies adhesion among cells expressing Npn1A and L1.1 to induce fasciculation. These experiments may reveal a novel mechanism(s) of semaphorin mediated guidance. To discover how growth cones of MLF axons translate Sema3D and TAG-1 into directed cell movements during fasciculation, in vivo timelapse imaging will be used to analyze growth cone motility in the absence of these guidance cues. From this work, a more thorough understanding of how guidance cues function to guide fasciculating axons may provide insight into molecular signaling defects underlying neural developmental diseases and potential approaches for facilitating axon regeneration following injury or disease. [unreadable] [unreadable]